Laccases (EC 1.10.3.2) are enzymes having a wide taxonomic distribution and belonging to the group of multicopper oxidases. Laccases are eco-friendly catalysts that use molecular oxygen from air to oxidize various phenolic and non-phenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and produce water as the only side-product. These natural “green” catalysts are used for diverse industrial applications including the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, and used as bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. Another large proposed application area of laccases is biomass pretreatment in biofuel and in the pulp and paper industries.
Laccase molecules are usually monomers consisting of three consecutively connected cupredoxin-like domains twisted in a tight globule. The active site of laccases contains four copper ions: a mononuclear “blue” copper ion (T1 site) and a three-nuclear copper cluster (T2/T3 site) consisting of one T2 copper ion and two T3 copper ions.
Laccases may be isolated from different sources such as plants, fungi or bacteria and are very diverse in primary sequences. However, they have some conserved regions in the sequences and certain common features in their three-dimensional structures. A comparison of sequences of more than 100 laccases has revealed four short conservative regions (no longer than 10 aa each), which are specific for all laccases.[7, 8] One cysteine and ten histidine residues form a ligand environment of copper ions of the laccase active site present in these four conservative amino acid sequences.
The best studied bacterial laccase is CotA laccase. CotA is a component of the outer coat layers of bacillus endospore. It is a 65-kDa protein encoded by the CotA gene.[1]
CotA belongs to a diverse group of multi-copper “blue” oxidases that includes the laccases. This protein demonstrates high thermostability, and resistance to various hazardous elements in accordance with the survival abilities of the endospore.
Recombinant protein expression in easily cultivatable hosts can allow higher productivity in a shorter time and reduces the costs of production. The versatility and scaling-up possibilities of the recombinant protein production opened up new commercial opportunities for their industrial uses. Moreover, protein production from pathogenic or toxin-producing species can take advantage of safer or even GRAS (generally recognized as safe) microbial hosts. In addition, protein engineering can be employed to improve the stability, activity and/or specificity of an enzyme; thus, tailor made enzymes can be produced to suit the requirement of the users or of the process.
Enzyme productivity can be increased by the use of multiple gene copies, strong promoters and efficient signal sequences, properly designed to address proteins to the extracellular medium, thus simplifying downstream processing.
Recombinant protein yield in bacterial hosts is often limited by the inability of the protein to fold into correct 3D-structure upon biosynthesis of the polypeptide chain. This may cause exposure of hydrophobic patches on the surface of the protein globule and result in protein aggregation. Mechanisms of heterologous protein folding in vivo are poorly understood, and foldability of different proteins in bacteria is unpredictable.
Yield of soluble active protein can sometimes be improved by changing cultivation conditions. In addition, there are examples when protein yield was improved by introducing single-point mutations in the protein sequence. However, no rationale has been identified behind finding suitable mutations.
Heterologous expression of laccase in Escherichia coli has often been used as a strategy to get around the problem of obtaining laccases that are not easily producible in natural hosts. The recombinant expression of Bacillus subtilis CotA in E. coli has allowed its deep characterization, structure solving, and functional evolution.[1, 2, 3] However, very often, the production yield is low, due to a strong tendency of this enzyme to form aggregates that renders the protein irreversibly inactive.[4] This tendency has been attributed to the fact that, in nature, CotA laccase is integrated in a spore coat structure via interaction with other protein components, and it is likely that correct laccase folding is enhanced by interaction with other proteins. When this laccase is recombinantly expressed as an individual polypeptide, those supporting interactions are missing and many miss-folded proteins form aggregates in bacterial cells. When expressed in higher microorganisms such as yeast, misfolded laccase molecules are, for a large part, degraded.
There is a need in the art for means and methods for improving the yield of laccases in heterologous expression systems. This is particularly true for bacterial laccases, such as CotA laccases.